1. Field of the Invention
The present invention relates to medical treatment of mammals and more specifically to methods and medicaments for the control of neutrophil elastase and cathepsin G in mammals. Additionally, the present invention relates to methods of synthesis of 2-O-desulfated heparin, a medicament useful for the control of neutrophil elastase and cathepsin G in mammals.
2. Background Art
Activated neutrophils play an important role in a number of human and other mammalian diseases by releasing a number of oxidant chemicals and enzymes after migration into an affected organ. While oxidants, such as superoxide anion, hydrogen peroxide and hypochlorous acid are injurious by themselves, the major destructive elements produced by activated neutrophils are cationic proteases, the bulk of which consist of elastase and cathepsin G. When neutrophils release these proteases, tissue destruction occurs unless the proteases are neutralized by sufficient extracellular anti-proteinases such as .alpha.-1-anti-proteinase.
Individuals with an inherited deficiency of .alpha.-1-anti-proteinase suffer unimpeded proteolytic lung destruction over a lifetime, resulting ultimately in the development of pulmonary emphysema. Cigarette smoking causes the influx of activated leukocytes into the lung, with subsequent degranulation and/release of proteases. Cigarette derived oxidants also inactivate .alpha.-1-anti-proteinase by oxidizing an important methionine near the active site. Elastase delivered to the alveolar lung unit as a result of the influx due to cigarette smoking, concurrent with oxidative inactivation of .alpha.-1-anti-proteinase activity, produces an imbalance of protease/anti-proteinase activity that is thought to be a major cause of human emphysema from cigarette smoking.
When the imbalance occurs within the airway, chronic airway inflammation is the result, and neutrophil derived elastase and cathepsin G are thought important in the pathogenesis of chronic bronchitis. If the imbalance occurs within the pulmonary vasculature, the resulting microvascular injury causes lung edema formation. In this fashion the influx of activated leukocytes and release of elastase and other neutrophil proteases are major causes of lung injury in the Adult Respiratory Distress Syndrome. Neutrophil derived elastase is also an important cause of proteolytic lung destruction in cystic fibrosis, a disease characterized by intense mucopurulent bronchitis and some of the highest levels of elastase activity measured in any human disease.
Also, diseases such as myocardial infarction and stroke, caused by sudden loss of organ blood flow, followed by blood flow restoration (ischemia-reperfusion injury) are characterized by magnification of tissue destruction during the reperfusion phase when activated leukocytes rapidly invade the already injured tissue. Neutrophil elastase delivered to ischemic reperfused organs has been demonstrated to play a pivotal role in the pathogenesis of reperfusion injury of the myocardium, bowel and other tissues. The role of cathepsin G in the processes above is not as well studied, but may be equally important, since there is twice as much cathepsin G present in the neutrophil as elastase.
Because elastase and cathepsin G are mediators of a variety of important human diseases, developing effective inhibitors of these enzymes is an active goal in experimental pharmacology. However, to date, no completely effective and safe inhibitor of both elastase and cathepsin G has been reported. A small organic inhibitor of elastase has been developed (C. P. Sommerhoff, et al., European Journal of Pharmacology (1991) 193:153-158), but it failed to demonstrate activity against cathepsin G. Two biomolecules, .alpha.-1-anti-proteinase inhibitor and bronchial secretory inhibitor, are sensitive to inactivation by neutrophil oxidants and are not likely to be effective in biologic environments where neutrophil oxidants and proteases are present simultaneously (D. C. Flenley, Quarterly Journal of Medicine (1986) 61:901-909; C. Vogelmeier, et al., Journal of Clinical Investigation (1991) 87:482-488). An inhibitor is needed which is simultaneously effective against both elastase and cathepsin G but is impervious to either proteolytic or oxidative inactivation. The sulfated polysaccharides have each of those desirable qualities.
It has been previously reported that heparin and other sulfated polysaccharides are potent non-competitive inhibitors of elastase and cathepsin G from human polymorphonuclear leukocytes (N. V. Rao, et al., A. M. Rev. Respir. Dis. (1990) 142:407-412; A. Baichi, et al., Biochem. Pharmacol (1980) 29:1723-1727; A. Baichi, et al., Biochem. Pharmacol. (1981) 30:703-708; K. Marossy, Biochim. Biophys. Acta, (1981) 659:351-361; A. Baichi, et al., Chem-Biol Interactions (1984) 51:1-11; A. Lutini, et al., Biochem. Int. (1985) 10:221-232; F. Redini, et al., Biochem. J. (1988) 252:515-519; and F. Redini, et al., Biochem. Pharmacol. (1988) 37:4257-4261.) It is believed that the basis for inhibition is by formation of electrostatic bonds between the negatively charged sulfate groups of the polysaccharide and the positively charged guanidinium groups of the arginine residues located at the surface of those highly basic enzymes such as elastase or cathepsin G. The interaction does not influence the active center of the enzyme but causes an indirect loss of elastolytic activity.
Of all the sulfated polysaccharides, heparin has the longest and safest history of use in man. From a toxicologic consideration, heparin is the most desirable inhibitor of elastase and cathepsin G but for the fact that it is an anti-coagulant even when delivered selectively to the lung by aerosol. It is believed that heparin acts as an anti-coagulant because of a repeated sequence of saccharides which binds specifically to the plasma protein anti-thrombin III, dramatically accelerating the rate at which anti-thrombin inhibits the procoagulant effect of thrombin on the cascade of blood coagulation. Only a portion of commercial heparin binds to anti-thrombin III, and passage of heparin over an affinity column of anti-thrombin III-sepharose removes the anti-coagulant fraction leaving an incompletely snlfated firaction devoid of anti-coagulant activity. However, utilizing this process to rid heparin of its anti-coagulant activity is too inefficient to be undertaken on a commercially practical scale.
Previously, it has been noted that the activity of a polysaccharide as an inhibitor of human polymorphonuclear leucocyte elastase (HLE) and cathepsin G is directly dependent upon the presence of intact sulfate groups. Dextran sulfate is a potent inhibitor of elastase, but non-sulfated dextran is not. Furthermore, the available literature suggests that even partial desulfation of polysaccharides eliminates inhibitory activity toward HLE and cathepsin G while chemical oversulfation enhances inhibitory activity. The importance of sulfate groups was studied using fragments of heparin obtained by chemical depolymerization with HNO.sub.2 followed by gel filtration (F. Redini, et al., Biochem. J., (1988) 252:515-519). Unmodified heparin fragments obtained by this latter process were potent inhibitors of elastase but retained their strong anticoagulant power. Increasing the degree of sulfation by chemical 0-sulfation of the fragments markedly increased their potency as elastase inhibitors but did not materially alter the anticoagulant activity of the fragments. On the other hand, N-desulfation followed by N-acetylation (to cover the remaining positive charge and reduce the anticoagulant activity of the fragments) completely eliminated inhibitory activity toward human leukocyte elastase and cathepsin G (Redini et al.). Chemical over-O-sulfation of the N-desulfated fragments not only restored inhibitory activity but gave the fragments higher inhibitory potential compared to their original counterparts with a similar degree of sulfation but containing N-sulfate groups. It has been suggested that not only was the degree of sulfation important to inhibitory activity, but that the presence of O-sulfates were more important than the presence of N-sulfates (Redini et al.). However, none of these highly effective, modified heparins were suitable for use in mammals due to their potent continuing anticoagulant activity.
Several chemical methods exist for inactivating heparin as an anti-coagulant. Most are based on techniques of chemical desulfation, since it is well established that degree of sulfation is an important determinant of anticoagulant activity.
N-desulfation by treatment of the pyridinium heparin salt with dimethylsulfoxide (DMSO) in five percent methanol for 1.5 hours at 50.degree. C. and total desulfation by similar treatment in DMSO in 10% methanol for 18 hours at 100.degree. C. are commonly used chemical modifications to remove anti-coagulant activity from heparin. Another method to remove anti-coagulant activity from heparin is acid hydrolysis at 55.degree.-60.degree. C. for 72 hours to produce partial N-desulfation. However, removal of all sulfates or even a partial desulfation by removal of N-sulfates inactivates heparin and other sulfated polysaccharides as inhibitors of human elastase and cathepsin G. Thus, the art teaches that currently utilized desulfation methods which remove anti-coagulant activity of heparin also destroy its ability to inhibit cationic leukocyte proteases such as elastase and cathepsin G.
Moreover, the art demonstrates that over-sulfation leads to increased activity against elastase and cathepsin G with continuing anticoagulant activity while desulfation leads to decreased anticoagulant activity with greatly diminished activity against elastase and cathepsin G. In contrast to what would be predicted by the prior art, the present invention provides that selective 2-O-desulfation of .alpha.-L-iduronic acid-2-sulfate eliminates the anticoagulant activity of heparin without destroying the activity of the modified heparin as an inhibitor of elastase and cathepsin G.
Heparin has been widely used as a blood anticoagulant. However, it has also been widely recognized that there is a lack of uniform anticoagulant activity among different heparins (M. Jaseja et al., "Novel regio- and stereoselective modifications of heparin in alkaline solution. Nuclear magnetic resonance spectroscopic evidence," Can. J. Chem., 67:1449-1456 (1989)). Therefore, studies have been made of the anticoagulant activity of heparin (Jaseja et al. and R. Rej et al., "Importance for Blood Anticoagulant activity of a 2-Sulfate Group on L-Iduronic Acid Residues in Heparin," Thrombosis and Hemostasis, 61(3):540 (1989). Both references, in studying variations of heparin disclose the preparation of a previously unreported compound, 2-O-desulfated heparin. Briefly, the Rej et al. and Jaseja et al. method starts with a solution of heparin in 0.1N sodium hydroxide which is then lyophilized, thereby effecting a selective displacement of the 2-sulfate group of .alpha.-L-iduronic acid 2-sulfate and leaving a 2-O-desulfated .alpha.-L-iduronic acid residue. This compound was shown to have minimal anticoagulant activity. No suggestion of elastase or cathepsin G inhibition or any uses requiring elastase or cathepsin G inhibition were made in these studies. Therefore, Rej et al. and Jaseja et al. found no activity for 2-O-desulfated heparin and thus did not disclose any effective doses for the compound for any use.
In their report of the chemistry of removing 2-O-sulfate from alpha-L-iduronic acid 2-sulfate, Jaseja et al., describe that alpha-L-iduronic acid 2-sulfate is converted to alpha-L-iduronic acid in a two-step process. First, selective displacement of the 2-O-sulfate group occurs, with formation of a 2,3-anhydro intermediate. The 2,3-anhydro intermediate is then further hydrolyzed to alpha-L-iduronic acid. Specifically, Jaseja et al., teach that 40 mg of beef lung heparin was added to 10 ml water. When this 0.4% solution was alkalinized with 0.1N sodium hydroxide to pH 11.2 to 11.5 and lyophilized, the 2,3-anydro intermediate was quantitatively formed. By their methods, when the pH of the solution was raised further to 12.5 to 12.8, further hydrolysis of the intermediate occurred, with quantitative formation of desulfated alpha-L-iduronic acid. Jaseja et al., demonstrate that diminished antithrombin III binding affinity is conferred by loss of the 2-O-sulfate, since both the 2,3-anhydro intermediate and fully hydrolyzed alpha-L-iduronic acid modifications had substantially decreased anticoagulant activity compared to the starting heparin. Thus, Jaseja et al., provides explicit instructions for a laboratory scale reaction to produce small quantities of 2-O-desulfated heparin by alkaline hydrolysis during lyophilization.
The present invention involves the surprising discovery that 2-O-desulfated heparin has elastase and cathepsin G inhibition activity. This activity was unexpected since, as discussed above, prior desulfation attempts that resulted in a decreased anticoagulant activity also resulted in a lack of elastase and cathepsin G inhibition activity. Thus, the novel use of 2-O-desulfated heparin to inhibit elastase and cathepsin G provides a solution to a long-felt problem in the art.
Additionally, applicants also unexpectedly discovered that, when the method of Jaseja et al. was performed on a commercially feasible scale, the compounds thus produced possessed significantly less elastase inhibitory activity then the compounds produced by their published small-scale method. The present invention therefore provides an effective method of producing 2-O-desulfated heparin in commercially useful quantities.